Autophagy is the cell's primary mechanism to degrade damaged organelles and cytoplasm and to protect itself from toxic protein aggregates not degradable by the proteasome. We will address new leads revealing induction and impairment of autophagy in Alzheimer's Disease (AD), identifying its role in Abeta generation, and showing for a requirement for presenilin (PS1) in autophagy function. The hypothesis that autophagy is impaired in AD will be investigated in human brain and FAD fibroblasts, cell lines, and mouse models of AD pathology (Aims 1 and 2). Novel antibodies marking specific compartments of the autophagic pathway and other organelles will be used in double-immunofluorescence and ultrastructural studies (including immunogold), along with other state-of-the-art techniques, to define the nature and significance of autophagy dysfunction in AD. The roles of PS1, PS2, and g-secretase activity in modulating autophagy will be determined and the basis for the observed selective impairment in autophagy-mediated protein degradation caused by FAD-related PS1 mutations will be investigated (Aim 2). We will also investigate the basis for distinctive autophagy abnormalities in Trisomy 21 (Down syndrome) and test the hypothesis that FAD-related APP mutations (Aim 3) also impair autophagy. Autophagy as a neuroprotective mechanism will be studied in primary neurons from normal mice, AD mouse models, and a novel transgenic mouse expressing GFP-tagged LC3, a protein associated with autophagy induction, which will enable real-time visualization of autophagy and will facilitate in vivo studies of autophagy modulation (Aim 4). As a prelude to modulating autophagy as a therapeutic intervention in neurodegenerative disease, we will investigate strategies to manipulate autophagy in the brain in vivo (Aim 4). These ongoing studies, the first to investigate autophagy systematically in AD, will provide a comprehensive assessment of the impact of autophagy and autophagy dysfunction and identify novel approaches to treat proteinopathies, including AD.